1. Field of the Invention
The present invention relates to improved blood contact surfaces for use in apparatus such as in artificial blood vessels and other implantable appliances and methods for producing the blood contact surfaces.
2. Description of Related Art
Synthetic materials are widely used to replace diseased or damaged portions of the human cardiovascular system. The use of grafts composed of synthetics such as polyethylene terephthalate and expanded polytetrafluoroethylene (ePTFE), has provided successful results in the replacement of large vessels such as the aorta, iliac, or femoral arteries. The application of these same synthetics, however, to small diameter arteries such as in coronary artery bypass or peripheral arterial bypass beyond the popliteal artery often produces thrombosis despite patency-enhancing pharmacological measures. Other applications of synthetics in which clot formation is a frequent problem include the replacement of veins, heart valves, and the artificial heart. Tissue-based prosthetics provide similar performance to synthetics, having acceptable function in some applications, but in others, still having the complication of thrombus formation. These thrombotic problems limit the usefulness of both tissue-based and synthetic devices, particularly in more demanding applications. Consequently, a goal of researchers for many years has been to develop a blood contact surface that provides for the reduction or elimination of thrombosis.
Natural blood contact surfaces, such as those found within blood vessels, possess mechanisms that prevent blood from clotting during normal passage along the surface. In the case of a mammalian artery, the immediate blood contact surface comprises a layer of non-thrombogenic endothelial cells (ECs). Immediately external to the endothelial cell layer is the remainder of the intima: a subendothelial matrix layer of basement membrane and underlying glycoprotein-bearing extracellular matrix, and the internal elastic lamina. Surrounding the intima layer is the multilaminate media structure containing smooth muscle cells (SMCs) and elastin, and surrounding this media is the adventitia, the most external layer comprised of fibroblasts and connective tissue. Both the subendothelial layer and media are generally considered to be thrombogenic in nature in order to maintain hemostasis when the vascular system is injured.
Intact endothelial linings are considered to be non-thrombogenic unless damaged. Because of their blood-contacting location, endothelial cells have been thoroughly investigated with respect to their anti-thrombogenic function. Endothelial cells are known to synthesize or bind a number of substances with coagulation-inhibiting or fibrinolytic function including heparan sulfate/antithrombin III, dermatan sulfate/heparin cofactor II, thrombomodulin/protein C/protein S, prostacyclin and tissue-type plasminogen activator. Furthermore, segments of endothelium-bearing autologous vessel transplanted from one site to another in an individual to bypass diseased vessels exhibit an incidence of thrombosis substantially less than that of synthetics used in the same application. For these reasons, it has been assumed that endothelial cells are responsible for the non-thrombogenic activity of vessels. According to this assumption, synthetic blood contact devices capable of thrombo-resistance similar to the native vasculature would require a blood contact surface of endothelial cells.
Numerous attempts have been made to provide prosthetic surfaces, and specifically vascular grafts, that include or develop an endothelial cell lining. The overwhelming majority of these attempts have been carried out as an intraoperative cell-seeding procedure. Intraoperative cell-seeding typically involves harvesting endothelial cells from the recipient during the procedure and immediately seeding these collected cells onto a vascular graft that has been pretreated with a substrate to enhance endothelial cell attachment. Substrates frequently applied to the synthetic surface include preclotted blood taken from the patient or extracellular matrix proteins such as fibronecfin, collagen, or laminin, either singly or in combination. This approach was first reported by M. B. Herring et al. in "A single staged technique for seeding vascular grafts with autogenous endothelium," Surgery 84:498-504 (1978). In this procedure, autologous cells were seeded onto a preclotted DACRON.RTM. graft. These seeded grafts demonstrated a decreased thrombus formation compared to control grafts without endothelial cells. In spite of the improved initial adherence of the cells to the synthetic surface afforded through the use of these various substrata, the shear forces resulting from blood flow nevertheless leads to the loss of a substantial fraction of the applied cells.
In a variation of the above, U.S. Pat. No. 4,960,423 to Smith describes the use of elastin-derived peptides to enhance endothelial cell attachment. Some studies have used combinations of extracellular matrix molecules and cells to provide a substrate for endothelial cell attachment and growth. For example, U.S. Pat. Nos. 4,539,716 and 4,546,500 to Bell describe means by which endothelial cells are grown on a living smooth muscle cell-collagen lattice. In addition, U.S. Pat. No. 4,883,755 to Carabasi et al. describes a technical method for seeding endothelial cells onto damaged blood vessel surfaces.
Alternative means of growing endothelial cells on vascular grafts have also been reported. For example, the use of physical force to apply endothelial cells to graft surfaces is described in U.S. Pat. No. 5,037,378 to Muller et al. In another example, U.S. Pat. No. 4,804,382 to Turina and Bittman describe the application of endothelial cells to a semi-permeable membrane in which the pores are filled with aqueous gels to allow endothelial cell coverage.
Another approach has been the seeding of endothelial cells onto biologically-derived surfaces, including pericardium, cardiac valve leaflets, amnion, and arteries. These efforts appear to have originated with J. Hoch et al. in "In vitro endothelialization of an aldehyde-stabilized native vessel," J. Surg. Res. 44:545-554 (1988), where the authors attempted to grow endothelium on ficin-digested, dialdehyde stabilized, bovine artery. Human venous endothelial cells were found to adhere to and spread on the remnant collagen surface of these enzyme-digested grafts, but no implant studies were performed. J. Hoch et al. also investigated the growth of endothelium on human amnion, on live, mechanically-scraped human artery, and again on ficin-digested, tanned bovine artery. (J. Hoch et al., "Endothelial cell interactions with native surfaces," Ann. Vasc. Surg. 2:153-159 (1989)) Although endothelial cell adhesion was observed on these surfaces by 2 hours, the longterm persistence of ECs on these surfaces was not examined, and, as in the previous study, none of these endothelialized materials were implanted as vascular substitutes. P. A. Schneider et al. showed that endothelial cells could be successfully seeded onto the remaining collagenous surface of baboon vessels from which the intima was removed. (P. A. Schneider et al., "Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells," J. Vasc. Surg. 11:365-372 (1990)) S. G. Lalka et al. used detergent extraction of canine arteries to produce an ethanol-fixed acellular vascular matrix onto which human umbilical vein endothelial cells were successfully seeded in vitro. (S. G. Lalka et al., "Acellular vascular matrix: A natural endothelial cell substrate," Ann. Vasc. Surg. 2:108-117 (1989))
In a study similar to that of Hoch et al. in J. Surg. Res., supra, L. Bengtsson et al. successfully grew human venous endothelium on the luminal surface of devitalized vessel segments denuded of endothelium and subendothelium. (L. Bengtsson et al., "Lining of viable and nonviable allogeneic and xenogeneic cardiovascular tissue with cultured adult human venous endothelium," J. Thorac. Cardiovasc. Surg. 106:434-443 (1993))
It is emphasized that in none of the foregoing studies have attempts to provide endothelial linings to biological tissues been carried out on tissues with an intact subendothelium. Furthermore, in none of these attempts have the tissues been tested as chronic vascular substitutes following endothelialization.
Biological tissues destined for implant use are commonly subjected to a preservation treatment employing fixative agents such as glutaraldehyde, formaldehyde, dialdehyde starches, polyepoxy compounds, or alcohols. Glutaraldehyde is the most commonly used crosslinking agent because it provides reduced immunogenicity, excellent tissue preservation, and stability in the implant environment. The use of glutaraldehyde as an agent to produce articles derived from biological tissues suitable for implantation is specified in a number of U.S. Patents, including U.S. Pat. Nos. 3,562,820 to Braun, 3,966,401 to Hancock et al., 3,988,782 to Dardik, 4,050,893 to Hancock et al., 4,098,571 to Miyata et al., 4,323,358 to Lentz et al., and 4,378,224 to Nimni et al. Although glutaraldehyde-treated tissues have generally provided good implant outcome, it has been shown that unreacted, residual glutaraldehyde can result in the inhibition or death of cells grown in contact with the fixed tissue. Previous investigators (see, for example, L. Bengtsson et al. supra) have shown that growth of endothelium on fixed tissue can be inhibited by residual glutaraldehyde.
The problem of residual glutaraldehyde has been addressed by blocking the reactive site on the aldehyde groups. It is well known that residual or free aldehyde groups can be effectively blocked using compounds containing amino groups such as amino acids or proteins. Grimm et al. ("Glutaraldehyde affects biocompatibility of bioprosthetic heart valves," Surgery 111:74-78 (1992)) describe a postfixation treatment by which reactive aldehyde groups can be passivated. Endothelial cell-seeded, glutaraldehyde-fixed tissue showed uninhibited cell growth following a 48 hour exposure to 8% L-glutamic acid. Nashef et al., in U.S. Pat. No. 4,786,287, specify the use of solutions containing an excess of aldehyde-reactive amines to increase the rate of aldehyde diffusion from fixed tissue by maintaining a low concentration of free aldehyde. U.S. Pat. No. 4,553,974 to Dewanjee describes another method to prepare collagenous tissues for endothelialization using a surfactant-detergent treatment, followed by glutaraldehyde fixation and anti-calcification treatments. Although endothelium has been previously grown on tissue surfaces, the treatments employed, including ficin digestion, detergent treatment, and mechanical removal, each disrupt the subendothelial layer, resulting in increased thrombogenicity. Additionally, tissues such as pericardium or amnion do not, in the native state, possess the required subendothelial matrix.
Recent efforts to provide a substrate that supports endothelial cell linings on graft surfaces include the use of in vitro cultured extracellular matrix. A. Schneider et al. used corneal endothelial cells to produce extracellular matrix on ePTFE vascular grafts. (A. Schneider et al., "An improved method of endothelial seeding on small caliber prosthetic vascular grafts coated with natural extracellular matrix," Clin. Mat. 13:51-55 (1993)) After production of an extracellular matrix, these original cells were then removed using Triton X-100 and NH.sub.4 OH, and the tubes were seeded again with bovine aortic endothelium. This approach showed that endothelium could be successfully grown on the extracellular matrix lining the ePTFE grafts, however, no implant studies were performed. Another approach by Y. S. Lee et al. employed fetal human fibroblasts in culture to secrete extracellular matrix onto a polyurethane tube. The fibroblast cells were then removed by one of several methods and the remaining matrix seeded with human omental endothelial cells. This method resulted in a patent graft at 5 weeks after implantation into a rat aorta. (Y. S. Lee et al., "Endothelial cell seeding onto the extracellular matrix of fibroblasts for the development of a small diameter polyurethane vessel," ASAIO Journal 39:M740-M745 (1993))
A similar approach has also been taken by H. Miwa et al., "Development of a hierarchically structured hybrid vascular graft biomimicking natural arteries," ASAIO Journal 39:M273-77 (1993). In this case, smooth muscle cells are layered over a DACRON.RTM. graft in an applied artificial matrix of collagen type I and dermatan sulfate glycosaminoglycan. A layer of endothelial cells is then grown on the artificial matrix to serve as the blood contact surface.
Since the endothelium is the acknowledged source of the antithrombotic behavior of the normal vasculature, an endothelial lining has been widely regarded as the means by which improved antithrombogenicity of cardiovascular implants will be achieved. Actual testing however, has provided mixed outcomes. A number of studies have been conducted in animals, with some reporting clear improvements in patency as a consequence of endothelialization. Several studies, though, report equivocal outcomes with little measurable improvement. (P. Zilla et al., "The endothelium: A key to the future," J. Card. Surg. 8:32-60 (1993)) P. Ortenwall et al., for example, showed similar patency outcomes between seeded and control grafts in both sheep and dogs. (P. Ortenwall et al., "Seeding of ePTFE carotid interposition grafts in sheep and dogs: species dependent results," Surgery 103:199-205 (1988))
Although the collective results of experimental seeding studies conducted in animals are suggestive of a performance improvement due to the addition of recipient endothelial cells, the data do not indicate a performance improvement to the level expected with the use of the autologous vessel. For example, in a test conducted in sheep, N. L. James et al. report that only one of six (1/6) endothelial cell seeded grafts was patent in comparison to six of six (6/6) autologous artery grafts. (N. L. James et al., "In vivo patency of endothelial cell-lined expanded polytetrafluoroethylene prostheses in an ovine model," Artif. Org. 16:346-53 (1992)) Similarly, application of the above described endothelialization methods to synthetic vascular implants in humans has shown little demonstrable improvement in patency, despite the observation that grafts seeded with recipient cells become endothelialized, at least in some cases. (P. Zilla et al., "The endothelium: A key to the future," J. Card. Surg. 8:32-60 (1993)) Thus, the actual clinical outcome achieved with endothelial-lined synthetics has not met the expectations raised by animal studies.